Variation in Physiological Indicators in Bathymodiolus Azoricus

Marine Environmental Research 70 (2010) 264e271

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Marine Environmental Research

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Variation in physiological indicators in Bathymodiolus azoricus (Bivalvia: Mytilidae) at the Menez Gwen Mid-Atlantic Ridge deep-sea hydrothermal vent site within a year

Virginie Riou a,b,*, Sébastien Duperron c, Sébastien Halary c, Frank Dehairs a, Steven Bouillon a,d, Inès Martins b, Ana Colaço b, Ricardo Serrão Santos b a Department of Analytical and Environmental Chemistry & Earth System Sciences, Vrije Universiteit Brussel, Brussels, Belgium b Department of Oceanography and Fisheries, IMAR-University of Azores, Horta, Portugal c Université Pierre et Marie Curie, UMR 7138 Systématique Adaptation Evolution, Paris, France d Department of Earth and Environmental Sciences, Katholieke Universiteit Leuven, Leuven, Belgium article info abstract

Article history: Bathymodiolus azoricus, thriving at Mid-Atlantic Ridge deep vents, benefits from a symbiosis with Received 3 March 2010 methane- and sulphide-oxidising (MOX and SOX) bacteria, and feeds on particulate and dissolved Received in revised form organic matter. To investigate the temporal evolution in their nutrition adult mussels were collected 11 May 2010 from one location at the Menez Gwen vent site (817 m depth) on four occasions between 2006 and 2007 Accepted 15 May 2010 and studied using different techniques, including stable isotope analyses and FISH. Gill and mantle tissues d13C and d15N signatures varied by 2e3& during the year and these variations were linked to Keywords: fluctuations in tissue condition index, C and N contents and SOX/MOX volume ratios as quantified by 3D- Deep ocean Hydrothermal vent FISH. October and January mussels presented a particularly poor condition, possibly related with the Mussel prolonged summer period of low sea-surface primary production and/or with the stress of the transplant Bathymodiolus azoricus to acoustically retrievable cages for the October mussels, and with their reproductive state in January Azores Triple junction mussels, since they were spawning. Our results point to the possibility that May mussels benefited from Food source a pulse of sinking sea-surface plankton material. Results underline the dependency of stable isotopic Stable isotopes signatures on the physiological state of the mussel at the time of collection, and on the type of tissue Condition index analyzed. Tissue analysis Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction turbulent plume mixing, and tidal cycles render the vent environment highly variable (Lalou et al., 1984; Chevaldonné et al., 1991). The rain of particles derived from sea-surface production is of Such variations may interfere when trying to detect the contribu- great significance in the flux of organic matter into the deep ocean, tion of photosynthesis-derived material on faunal density, distri- and is thought to form a labile food source used for reproductive bution, migration and reproductive cycle at vent sites. growth in many deep-sea fish, echinoderms and bivalves (Tyler, Nevertheless, surface-derived carbon biomarkers (fatty acids) were 1988). However, at deep-sea hydrothermal vents and cold seeps detected in the tissue lipids of Bathymodiolus mytilids showing low (>200 m), the influence of sea-surface photosynthesis-derived numbers of bacterial symbionts, thriving as deep as 2600 m at 13N primary production may appear negligible because of high East Pacific Rise vents (Ben-Mlih et al., 1992). At shallower (650 m) autochthonous chemosynthetic primary production (Tarasov et al., cold seeps on the Louisiana Slope, ‘Bathymodiolus’ childressi was 2005),. In addition, variations in hydrothermal fluid discharge, also observed to supplement its nitrogen requirements by feeding selectively on nitrogen-rich bacterioplankton, as suggested by the variability in nitrogen isotope ratios (Pile and Young, 1999). * Corresponding author at: ANCH-VUB, Pleinlaan 2, 1040 Brussels, Belgium. Tel.: Bathymodiolus mussels are the dominant fauna of numerous þ32 26293970; fax: þ32 26293274. . seep and hydrothermal vent ecosystems (Van Dover et al., 2002) E-mail addresses: [email protected] (V. Riou), [email protected] and mussel beds act as habitat or substrate for numerous meio- (S. Duperron), [email protected] (S. Halary), [email protected] (F. Dehairs), [email protected] (S. Bouillon), [email protected] (A. fauna species (Van Dover and Trask, 2000). At the Azores Triple Martins), [email protected] (A. Colaço), [email protected] (R. Serrão Santos). Junction on the Mid-Atlantic Ridge (MAR), the Menez Gwen (MG)

0141-1136/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.marenvres.2010.05.008 V. Riou et al. / Marine Environmental Research 70 (2010) 264e271 265 and Lucky Strike (LS) hydrothermal vent sites ecosystems are and Pond et al. (1998) detected phytoplankton biomarkers in B. dominated by Bathymodiolus azoricus beds (Desbruyères et al., azoricus body lipids, indicating that phytoplankton is accessible for 2000, 2001) which usually are clustering at short distance from the deep hydrothermal vent ecosystem and can be assimilated by the vents (Sarradin et al., 1999; Colaço et al., 1998) but may stretch the vent mussel. Colaço et al. (2009) observed an increase in as far as 3 m away from the hot fluids at the LS Eiffel Tower site phytoplanktonic biomarkers relative to other phospholipid fatty (Cuvelier et al., 2009). Ecological studies of deep-sea hydrothermal acids for MG specimens, which coincided with the autumn bloom. vent habitats are complicated because of the location (mid-ocean) B. azoricus activity at MG thus seems to be influenced, at least to and depth (800e4000 m) of the vent sites. Early biological studies some degree, by surface primary production. on living vent fauna were often restricted to shipboard studies Deposition of phytodetritus to the seafloor is a seasonal, performed on specimens recovered using either a deep-sea possibly pulsed, process and its significance for B. azoricus nutrition submersible or a remote operating vehicle (ROV). These devices can is difficult to assess from annual samplings. In addition, the only operate under good weather conditions, generally during the detection of phytoplankton-derived biomarkers might be masked summer months (from June to September) at the Azores Triple by a changing reproduction-related physiological state of the Junction. The restricted sampling window corresponds to episodes mussel. To resolve these different interfering processes we decided of low surface primary production in the Azores region, where high to investigate the effects of a variable supply of surface-derived photosynthetic production generally occurs between November food on the mussel’s physiology, by collecting vent mussels at 2e3 and May (Colaço et al., 2009). This may induce bias and to under- months intervals covering a complete yearly cycle. Several indica- estimating the impact of photosynthetic production on deep-sea tors can help document the physiological state of the vent mussel. animals including B. azoricus. Time series, or at least sampling of The allometric tissue condition index (TCI: soft tissue dry weight/ a given site several times a year, are necessary to track seasonal shell internal volume) has been recognized as a suitable proxy for phenomena. In this regard, acoustically retrievable cages were physiological condition of the mussel (e.g. Voets et al., 2006). A high developed. Once positioned and filled with surrounding mussels TCI reflects a healthy physiological condition, while a decreased using a ROV these cages are acoustically released and once energy uptake and an increased maintenance cost (for survival) can surfacing, can be recovered from a small research vessel. This result in a low TCI, revealing reduced growth and/or reproduction technique enables faster, year-round and ROV-independent mussel (De Coen and Janssen, 2003). A time-course examination of TCI collection (Fig. 1, Dixon et al., 2001). should thus inform on the health condition of the mussels, as At known northern MAR vent sites, B. azoricus occurs at possibly influenced by their translocation and adaptation to the different depths ranging from 817 to 3080 m (Menez Gwen -MG-, cage environment, variability in food supply and spawning events. Lucky Strike, Rainbow and Broken Spur, Desbruyères et al., 2000; Since tissue C:N ratio can be affected by the storage or use of O’Mullan et al., 2001). B. azoricus possesses a dual endosymbiosis reserves, it also represents a powerful indicator of the animal’s with methane- and sulphide-oxidising Gammaproteobacteria physiological condition (Okumura et al., 2002). Indeed, proteins (MOX and SOX, respectively, Fiala-Médioni et al., 2002; Duperron and nucleic acids are enriched in nitrogen, compared to the et al., 2006) located in specialised cells (bacteriocytes) of the gill nitrogen-poor lipids and carbohydrates (e.g. glycogen), also used as epithelial tissue. These symbionts ensure (at least part of) the host storage compounds. The catabolism of lipids and carbohydrates nutrition (Fisher, 1990; Martins et al., 2008; Riou et al., 2008) with will lead to a decrease in C:N ratio, and proteins will only be used as assimilation of particulate and dissolved organic matter demon- an internal energy source when glycogen reserves are exhausted, strated to represent a further nutritional mode to B. azoricus (Riou resulting in increased nitrogen excretion relative to respiration and et al., 2010). increasing C:N ratio (Gabbott, 1983). Dixon et al. (2006) report for MG vent mussels that the amount In addition to monitoring mussel physiological indicators, we of storage tissue in the mantle decreases when the mussels are assessed the relative importance of MOX and SOX symbionts to B. producing gonads. They also observed a decreased digestive gland azoricus nutrition by the relative volumes they occupy in gill bac- activity coinciding with low surface ocean chlorophyll a concen- teriocytes (Halary et al., 2008), and we investigated the possibility trations (indicative of photosynthetic activity). Colaço et al. (2009) to trace the signal of surface ocean production in the mussel’s bulk tissues isotopic signatures. Stable isotopic signatures are widely used to document food sources and trophic inter-dependencies (e. g. Van Dover and Fry, 1989; Colaço et al., 2002; Bergquist et al., 2007; Soto, 2009), and have been very useful in resolving the contributions of seep depleted methane and photosynthetic carbon to seep and vent fauna (e.g. Levin and Michener, 2002; Levin et al., 2009). Indeed, the proportion of 15N/14Nor13C/12C in a tissue will be modified only if certain types of metabolites are excreted or if C and N from a food source with different d13C and d15N signatures are incorporated. Carbon isotope analyses helped distinguishing hydrocarbon seep animals harbouring SOX symbionts (40

MOX symbiont (Duperron et al., 2008), the Cy5-labelled BangT-642 probe specific for the SOX symbiont (Duperron et al., 2005) and the additional Cy3-labelled general Eubacteria probe EUB338 (Amann et al., 1990)asinHalary et al. (2008). Three-dimensional fluores- cence microscopy was performed using a BX61 microscope (Olympus Optical Co., Tokyo, Japan) and following the procedure established by Halary et al. (2008), for image acquisition and treatment with the ‘SymbiontJ’ software. Briefly, image stacks were obtained by acquiring images on consecutive focal planes separated by 0.3 mm over the whole thickness of the sections using a 60 objective (NA 1.30). Three stacks, one for each fluorochrome, were produced from each part of the tissue section examined: (i) the green channel corresponded to the ImedM probe (MOX); (ii) the blue channel to the BangT probe (SOX); and (iii) the red channel to the EUB338 probe. This last channel was binarized and used as a mask when processing the 2 other channels, such that MOX appear in yellow and SOX in purple (Fig. 2). Bacteriocytes were isolated from the whole image, the corresponding stacks were segmented by binarization and the number of voxels belonging to the bacterial objects was computed. Total volume occupied by bacteria and respective proportions of each phylotype were computed for every bacteriocyte.

3. Results

3.1. Mussel condition

The TCI for mussels collected in October 2006 was significantly lower than for the August 2006, January and May 2007 mussels (KruskaleWallis multiple sample comparison KeW, H(3, N ¼ 36) ¼ 17.2, p ¼ 0.0006, Fig. 3A). The TCI values for the January mussels were also significantly lower than those for May mussels (ManneWhitney pairwise comparison M-W, p ¼ 0.03). Shell volume normalized gill weight was significantly lighter for October mussels, compared to the other collections all presenting similar normalized gill weights (KeW, H (3, N ¼ 40) ¼ 15.1 p ¼ 0.0018, Fig. 3B). Shell volume normalized mantle tissue dry weight for the January and October mussels was lower than for the August and May mussels (KeW, H(3, N ¼ 40) ¼ 22.9, p ¼ 0.0000, Fig. 3C).

3.2. Tissue C and N contents

Gill C:N atom ratios for October mussels were significantly smaller than for the other months (KeW, H(3,N ¼ 40) ¼ 21.3 p ¼ 0.0001, Fig. 4A). In general, gill C:N ratios were consistently lower than for the Fig. 3. Bathymodiolus azoricus total and specific tissues condition. A: total soft tissue mantle tissue, mainly due to higher N contents in gill tissue. Indeed, condition index (TCI), B: gill tissue dry weight normalized by the mussel shell volume, mantle N content remained almost constant at 8.8% over the year, C: mantle tissue dry weight normalized by the mussel shell volume, in August (N ¼ 10) while gill nitrogen content was consistently higher (around 9.4%). and October (N ¼ 10) 2006 and January (N ¼ 10) and May (N ¼ 10) 2007. Mantle C:N ratios in August specimens were particularly high (median C:N around 6), while the lowest ratios were measured in 3.3. Tissue C and N stable isotopic compositions October and January (median C:N around 5). This was due to 13 reduced carbon contents (38%) recorded in October and January Mantle and gill d C signatures were almost identical in August, mussels as opposed to the 41e45% contents measured in August October and May, but diverged in January, when gill tissues became 13 and May mussels. significantly enriched in C compared to the mantle (by þ2.6&;

Fig. 2. FISH detection of SOX and MOX symbionts in a Bathymodiolus azoricus gill filament. Preserved gill tissue section was hybridized with fluorescent 16S rDNA probes specificto the sulfide- and methane-oxydizing bacterial symbionts (SOX: purple and MOX: yellow, respectively) and the tissue section was observed with a 60 magnification. 268 V. Riou et al. / Marine Environmental Research 70 (2010) 264e271

Fig. 4. Bathymodiolus azoricus gill (ﬁlled symbols) and mantle tissues (empty symbols) C and N composition. A: molar C:N ratio, B: carbon content (%C) and nitrogen content (%N, N ¼ 10, medians; 25e75%).

MeW, p ¼ 0.029, Fig. 5A). Moreover gill and mantle tissues in vertical carbon flux above the vent. However, we are confident that October were significantly depleted in 13C relative to August and mussels in all cages were exposed to overall similar conditions prior May (by about 1.0 and 1.4&, respectively, KeW H (3,N ¼ 40) ¼ 20.2 to sampling. p ¼ 0.0002 for gill tissue and H (3,N ¼ 40) ¼ 15.2 p ¼ 0.0017 for Methane and sulfide concentrations up to 100 mM and 62 mM, mantle tissue). Gill d15N signatures overlapped with mantle tissues respectively, have been recorded over Menez Gwen mussel beds signatures for all 4 collections, but gill d15N signatures showed (Sarradin et al., 1998), and such concentrations would allow significant 15N enrichment for individuals collected in January and endosymbiont activity to sustain at least part of the carbon May, as compared to August and October collections (þ1.8 to 2.9&, requirements of the hosts (Martins et al., 2008). In addition, a peak KeW H(3,N ¼ 40) ¼ 24.6 p ¼ 0.0000). Mantle tissue was significantly in mass flux was recorded by a sediment trap moored at 1550 m, enriched in 15N only for October and May (þ1.9&,MeW p ¼ 0.003). 2 km away from the Rainbow vents, about one month after a surface ocean bloom evidenced by satellite observation 3.4. 3D-FISH (Khripounoff et al., 2008), revealing a close coupling between deep ocean carbon flux and surface production. Such a coupling is Three B. azoricus individuals from each cage were analyzed for expected to be even tighter for the shallower MG vent site the volumes occupied by SOX and MOX symbionts in the gill bac- (800e840 m), since temporal offset between surface production teriocytes. The average TCI, GI, C:N, d13C and d15N values of these and deep ocean flux is likely to be less in a shallow water column. sub-samples used for 3D-FISH fitted the median values based on Satellite observations of chlorophyll a (Chl a) in the Menez Gwen the 10 replicates. For each specimen 28 to 38 bacteriocytes were area revealed monthly concentrations of 0.098, 0.137, 0.189 and isolated and analyzed by the 3D-FISH SymbiontJ program (Halary 0.098 mg m 3 for August, October 2006, January and May 2007, et al., 2008). August and May mussels displayed identical SOX/ respectively (Colaço et al., 2009). January mussels were collected MOX ratios while the ratios for October were significantly higher when Chl a concentrations were highest. The 2007 spring bloom and those for January lower (distributions were not normal and we occurred exceptionally early in the year (right after the January cage used the non-parametric KeW statistical test: H (3, N ¼ 356) ¼ 73.0 collection) and was particularly short when compared to the situ- p ¼ 0.0000, Fig. 6). ation during the 10 years preceding our experiment (see Dixon et al., 2006; Colaço et al., 2009). 4. Discussion All measured physiological indicators displayed significant variation among specimens collected at the different times of the Since all specimens were collected from a small area (6 m2)itis year. In particular, specimens collected in October and January dis- probable that they belonged to a single population of mussels. It played marked differences. TCI values for October specimens were was not possible to in situ monitor the evolution of physico- indeed significantly lower than for other months (Fig. 3A), indicating chemical conditions and the sediment trap deployed in the a decreased energy uptake and/or increased maintenance cost for immediate vicinity of the cages failed impeding the monitoring of survival. Since nitrogen levels in gill and mantle tissues were in the

Fig. 5. Bathymodiolus azoricus gill and mantle tissues C and N stable isotopic signatures. Bulk gill (full diamonds) and mantle (empty diamonds) tissues carbon (A) and nitrogen (B) isotopic compositions (N ¼ 10, median; 25e75%). V. Riou et al. / Marine Environmental Research 70 (2010) 264e271 269

coastal M. edulis was observed to vary between 4 and 6 along an annual cycle with the lowest C contents (around 35%) associated with spawning periods (lowest glycogen contents, Rodhouse et al., 1984; Smaal and Vonck, 1997). That situation is also observed in B. azoricus collected in January. Indeed, compared to August and May mussels, the mantle tissue of January specimens was overall lighter (Fig. 3C) and displayed low C:N ratios and C contents (Fig. 4). The most striking difference for the January specimens was the 13C and 15N enrichment of the gill tissue. Gill and mantle enrichment in heavy nitrogen was also observed in May specimens. 13C enrichment could reflect a larger contribution of methane-derived carbon by MOX symbionts in gill tissues (Trask and Van Dover, 1999; Colaço et al., 2002; Salerno et al., 2005). Indeed, the SOX/ MOX ratio was lower in January compared to the other months, despite the fact that in January the gills appeared as developed as in Fig. 6. Relative volumes occupied by Bathymodiolus azoricus’ SOX and MOX symbionts. ¼ ¼ August and May (Fig. 3B). An alternative explanation for the Individual gill bacteriocytes from mussels collected in August (N 3 mussels, n 87 d13 d15 bacteriocytes, mean SE/SD) and October 2006 (N ¼ 3, n ¼ 89), January (N ¼ 3, n ¼ 97) enriched C and N signatures in January could be the assimi- and May 2007 (N ¼ 3, n ¼ 83) were analyzed by 3D-FISH as described in Material and lation of photosynthesis-derived carbon and nitrogen, linked to the Methods. observed higher Chl a concentration at the surface at the time of sampling. Indeed, pelagic particulate organic matter collected near same range as for the other months, we can conclude that B. azoricus the northern MAR hydrothermal vents had values around specimens collected in October were not starved, as they did not d13C ¼23& and d15N ¼þ3& (Riou et al., 2010). As opposed to the exploit protein as an internal energy source (Gabbott, 1983). The %C variations in stable isotopic composition linked to symbionts was lowest in gills of October specimens and in mantle tissues from dynamics and which can be detected instantaneously in the gill October and January specimens. This could be the consequence of tissue, the assimilation of sea surface-derived material with its the use of glycogen and lipid carbon-rich reserves for the production specific isotopic signature will eventually become detectable in the of energy in the October gill tissues. In addition, stable carbon isotopic signature of the mussel tissues after some delay isotopic signatures in gill and mantle were significantly more (Dattagupta et al., 2004). A considerable increase in sea-surface negative in October compared to all other months. Since lipids are primary production was ongoing above Menez Gwen since generally depleted in 13C compared to bulk tissue, proteins and November 2006 (thus 3 months before the January sampling) and carbohydrates (DeNiro and Epstein,1978), consumption of lipids for continued till March 2007 (i.e., 1.5 month before the May collection, energy production in case of food deprivation or stress, should lead Colaço et al., 2009) Significant tissue carbon turn-over would have to an increase in tissue d13C. This is not observed and B. azoricus’ occurred over a time span of 4.5 months (Dattagupta et al., 2004) tissues rather were depleted in both 15N and 13C(Fig. 5). Thus, and thus any assimilation of sea surface-derived material would in carbohydrate reserves, rather than lipid reserves, might have been principle be detectable in B. azoricus tissues. Finally, differences burnt, resulting in a decrease in 13C content. between C and N signatures in gill and mantle tissues for January However, according to earlier studies, more negative signatures specimens could result from higher particulate organic matter in MAR mussels can also reveal a larger contribution of SOX incorporation rates into the gill compared to mantle, as was clearly symbiont chemoautotrophy to the host carbon pool (e.g. Trask and observed during lab-based experiments (Riou et al., 2010). Van Dover, 1999; Colaço et al., 2002; Salerno et al., 2005; De Furthermore, the range of isotopic values measured for both C and Busserolles et al., 2009). This is congruent with our observation N was overall larger in gill compared to mantle tissue. of higher SOX/MOX ratios in October compared to the other months (Fig. 6). In case the abundances of bacteria remained constant over 5. Conclusion time, higher SOX/MOX ratios would reflect either an increase in SOX symbionts volumes or a decrease in MOX. The low TCI This study revealed differences for several physiological indica- measured for October points to a lower general condition of the tors among B. azoricus specimens collected at different periods of the mussel, which might be accompanied by overall symbiont loss. Gill year. Specimens collected in October displayed a lower tissue weights of October individuals were considerably lower compared condition index (TCI) and carbon content, linked to a poor condition. to the other months (Fig. 3B), indicating reduced gill size in October These mussels had apparently depleted their energy reserves, mussels. To summarize, the lower TCI and C content of October possibly as a result of the prolonged summer period of low sea- specimens could reflect stress (nutritional and/or linked with the surface primary production (assuming this surface ocean carbon transfer to cages). The “dominance” of SOX as suggested by SOX/ source was delivered to the mussels). It could also be that mussels MOX ratios and stable C isotopes could also be linked with higher still suffered stress due to the detachment from their initial substrate sulfide emissions, as was previously observed in aquarium exper- (breakage of byssus threads) and the transfer to cages where they iments (Halary et al., 2008). were kept at a greater distance from the substrate and fluid emis- TCI values for January were in the lower range of observed sions. The mussels might thus have had to adapt to reduced values but were not significantly different from values for October. hydrothermal flow (and thus to less substrate for the symbionts’ Gill C and N contents in January were similar to values observed in primary production). This adaptation, together with the occurrence August and May, while the January SOX/MOX ratios were lower of spawning events in January has limited the number of samples than those for August and May. Spawning was previously observed appropriate to follow genuine effects of variation in nutrition/ to occur during the January/February period for Menez Gwen feeding. Future nutrition studies should thus consider the deploy- mussels (e.g. Colaço et al., 2006) and January 2007 specimens kept ment of a higher number of cages, some of which could eventually be alive in the laboratory did spawn soon after transfer to the aquaria left for an extended duration on the seafloor before recovery. (Riou V. pers. obs.). Spawning activity in Mytilus edulis has also been Specimens collected in January displayed features possibly reported to lead to low TCI (Rodhouse et al., 1984). C:N ratio in wild reflecting their reproductive status and/or the influence of 270 V. Riou et al. / Marine Environmental Research 70 (2010) 264e271 photosynthesis-derived organic matter input. Collecting samples at Cuvelier, D., Sarrazin, J., Colaço, A., Copley, J., Desbruyères, D., Glover, A.G., a single location during different seasons over the year enables Tyler, P., Santos, R.S., 2009. Distribution and spatial variation of hydrothermal faunal assemblages at Lucky Strike (Mid-Atlantic Ridge) revealed visualizing the effect of external and internal processes that would by high-resolution video image analysis. Deep Sea Research I 56 (11), not be detected otherwise. A comparative study of specimens 2026e2040. sampled at different moments over the year could reveal the Dattagupta, S.D., Bergquist, C., Szalai, E.B., Macko, S.A., Fisher, C.R., 2004. Tissue carbon, nitrogen, and sulphur stable isotope turnover in transplanted Bathy- dynamics of mussel physiology in relation to food availability and modiolus childressi mussels: relation to growth and physiological condition. life cycle, and could possibly also reveal seasonal or cyclic patterns. Limnology and Oceanography 49, 1144e1151. These sampling periods should be chosen to match with specific De Busserolles, F., Sarrazin, J., Gauthier, O., Gélinas, Y., Fabri, M.C., Sarradin, P.-M., Desbruyères, D., 2009. Are spatial variations in the diets of hydrothermal fauna events such as phytoplankton blooms. To ascertain the links linked to local environmental conditions? Deep-Sea Research II 56, 1649e1664. between biotic and abiotic processes, monitoring the characteris- De Coen, W.M., Janssen, C.R., 2003. The missing biomarker link: relationships tics of in situ habitats and associated populations and increasing the between effects on the cellular energy allocation biomarker of toxicant-stressed fi Daphnia magna and corresponding population characteristics. Environmental number of sampling time points would be highly bene cial for our Toxicology and Chemistry 22 (7), 1632e1641. understanding of deep-sea hydrothermal vent ecosystems. This DeNiro, M.J., Epstein, S., 1978. Influence of diet on the distribution of carbon might become possible in the context of deep-sea marine obser- isotopes in animals. Geochimica et Cosmochimica Acta 42, 495e506. fl vatories which are currently being implemented. DeNiro, M.J., Epstein, S., 1981. In uence of diet on the distribution of nitrogen isotopes in animals. Geochimica et Cosmochimica Acta 45, 341 e351. Desbruyères, D., Almeida, A., Biscoito, M., Comtet, T., Khripounoff, A., Le Bris, N., Sarradin, P.-M., Segonzac, M., 2000. A review of the distribution of hydro- Acknowledgements thermal vent communities along the northern Mid-Atlantic Ridge: dispersal vs. environmental controls. Hydrobiologia 440, 201e216. We acknowledge the LabHorta team, A. Martins and I. Bash- Desbruyères, D., Biscoito, M., Caprais, J.C., Colaço, A., Comtet, T., Crassous, P., Fouquet, Y., Khripounoff, A., Le Bris, N., Olu, K., Riso, R., Sarradin, P.-M., machnikov for the experimental support. We thank the Victor 6000 Segonzac, M., Vangriesheim, A., 2001. Variations in deep-sea hydrothermal vent team, the crews of “R/V Arquipélago” and “Pourquoi Pas?” and the communities on the Mid-Atlantic Ridge near the Azores plateau. Deep-Sea chief scientists of the MoMARETO cruise, J. Sarrazin and P.-M. Sar- Research Part I 48 (5), 1325e1346. Dixon, D.R., Dando, P.R., Santos, R.S., Gwynn, J.P., the VENTOX Consortium, 2001. radin, who made the cages deployment and recovery possible. Retrievable cages open up new era in deep-sea vent research. InterRidge News Financial support for this research was provided by the EU projects 10, 21e23. MoMARNET-FP6-RTN/2003/505026 and the Research Foundation Dixon, D.R., Lowe, D.M., Miller, P.I., Villemin, G., Colaço, A., Santos, R.S., Dixon, L.R.J., Flanders (FWO-Vlaanderen, contract G.0632.06). The EU Frame- 2006. Evidence for seasonal reproduction in the Atlantic vent mussel Bathy- modiolus azoricus, and an apparent link to the timing of photosynthetic primary work Contract No EVK3-CT1999-00003 (VENTOX) funded the cage production. Journal of the Marine Biolological Association of the United system. IMAR-DOP/UAç research activities are additionally sup- Kingdom 86, 1363e1371. ported through the pluri-annual and programmatic funding Duperron, S., Nadalig, T., Caprais, J.C., Sibuet, M., Fiala-Médioni, A., Amann, R., Dubilier, N., 2005. Dual symbiosis in a Bathymodiolus sp. Mussel from schemes of FCT (Portugal) and Azorean Regional Directorate for a methane seep on the Gabon Continental Margin (Southeast Atlantic): 16S Science and Technology (DRCT, Azores, Portugal) as Research Unit rRNA Phylogeny and distribution of the symbionts in gills. Applied and Envi- e #531 and Associate Laboratory #9. This work has been conducted ronmental Microbiology 71 (4), 1694 1700. Duperron, S., Bergin, C., Zielinski, F., Blazejak, A., Pernthaler, A., McKiness, Z.P., in accordance with institutional, national and international guide- DeChaine, E., Cavanaugh, C.M., Dubilier, N., 2006. 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